Multiplex PCR allows amplification of multiple targets in a single PCR experiment. It is possible to amplify several sections of a single template, or to amplify different templates using a number of primer sets. If there are multiple primers in a reaction, it can be difficult optimise the PCR reaction to maximise the efficiency of every primer, and it is likely that some cross-hybridisation and mis-priming will occur.

Figure 3B from Wen and Zhange (2012). A comparison of multiplex PCR (Lanes 1-4) and universal multiplex PCR (lanes 5-8), using the same primers with universal adaptors. The band intensity from traditional PCR is very variable, but it is consistently strong when the universal adaptors are used.

Image credit: BioMed Central

The Method

Wen and Zhang from Shandong Agricultural University have devised a way around the inconveniences of multiplex PCR to develop a universal multiplex PCR method. ‘Universal adaptors’ are linked to specific primers, making the annealing temperature of the adaptor-primer structures 70°C. (more…)

In plant science, many published papers involve at least one loss-of-function mutant. A huge number of mutant Arabidopsis lines exist in labs all around the world, detailed in as many journal articles. Now however the genotype and phenotype information for loss-of-function Arabidopsis mutants is stored one place: a dataset assembled by Johnny Lloyd and David Meinke of Oklahoma State University.

Lloyd and Meinke painstakingly went through TAIR, their own database SeedGenes.org, and PubMed to find 2400 Arabidopsis thaliana genes with a loss-of-function mutant phenotype. Out of necessity, the database excludes the effects of under- or over- expression of genes.

The phenotypic effects of gene knock-outs were classified into four groups: essential, morphological, cellular-biochemical, and conditional. The groups were divided into classes reflecting the developmental stage or organ where the phenotype manifests itself, and further divided into subsets which specify the characteristic affected by the phenotype, for example ‘pigmentation’, ‘gamerophyte defective’, and ‘stomata, trichomes’.

The dataset is found in the supplementary data of the paper. Supplemental Table 2 is the complete dataset. On tab 1 the dataset is sorted by locus number and includes 19 columns of information on the gene and the mutant phenotype. This information encompasses the classification of the phenotype, a description of the phenotype, and a reference to the lab in which the research was carried out. (more…)

—- This page was updated on 17 and 21 August 2012 with recommendations from @BMC_series and others. I will make further changes if necessary so please contact me if you have any suggestions —-

When reviewing the recent GARNet workshop Making Data Accessible to All, we thought it was a good idea to collate the important bioscience databases on the web. I set to work and came up with the table below.

GARNet are keen to get an impression of how the plant science community actually use online databases, so please use the form at the end of this post to let us know how you use these types of resources. Do you deposit data in them, use them to guide practical work, or build whole research projects around them?

You’ll notice there are some blank spaces – if you know a resource that can fill it, please let me know. Likewise if you think I’ve got something wrong (I have personally worked with only a handful of these resources!) or missed off your favourite database, please leave a comment, tweet me or use the form below to tell me about it. (more…)

Xanthomonas spp. are plant pathogens that modulate their host’s gene expression in order to facilitate infection. They do this using transcription activator-like effectors (TALEs). Two domains are conserved in TALEs: an N-terminus, required for type III secretion into the plant cells; and a C-terminus with transcription factor activity. In the middle is a set of tandem repeats of amino acids, which mediates binding to host DNA.

As the binding and effector domains of TALEs can be customised, the possibility of using them for molecular and synthetic biology has been explored for some time. They have been used to change gene expression in plants, yeast, and even human cells.

TALEs have been adapted by researchers to make TALE nucleases (TALENs) – TALEs attached to a FOK1 nuclease domain. TALENs work in pairs that flank either side of the target site so that the nuclease domains meet at the point of cleavage. The nucleases cause a double-stranded DNA break, which is fixed imperfectly, causing an insertion or deletions.

In May this year, a paper was published demonstrating the huge impact TALEs could have on agriculture. Li et al. prove that transcription activator-like effector nucleases (TALENs) can be used to render rice resistant to the major agricultural pathogen, Xanthomonas oryzae pv. Oryzae (Xoo). (more…)

Kun-hsiang Liu, Matthew McCormack and Jen Sheen from Harvard have developed a PCR-based method of identifying mutations in Arabidopsis. It is cheaper and easier than traditional methods of identifying mutations, using bench-top PCR and a new user-friendly method of bioinformatics analysis using web-based resource Galaxy. Liu et al. estimate that using their method to identify a mutation mapped to a 550kb genomic region will cost roughly US$500, a fraction of the usual ten thousand dollar cost of currently used methods of mutant identification.

Liu et al. tested the new method of identifying mutations by searching for new nitrogen response genes. They generated an Arabidopsis thaliana line in which LUCIFERASE was driven by the promoter for nitrogen response marker NIR. Using EMS-mutagenesis, the team made 25 000 mutant NIR:LUC lines and identified seedlings that were nitrate insensitive (nis) or showed nitrate constitutive response (ncr).

When the lines were made, the phenotypes were identified and a second generation was grown. Three second generation lines – ncr1, nis1 and nis2 – were selected for further investigation.

Liu et al. used their novel TPSeq method to locate the mutations causing the ncr1, nis1 and nis2 phenotypes. (more…)

A current paper in Plant Methods assessed the pros and cons of two RNA labeling methods for AGRONOMICS1 tiling arrays, concluding that random priming is more suitable for organelle transcriptome analysis as it can label non-polyadenylated transcripts effectively. They also generated new TAIR-10 based CDF files, which can be used to re-analyse existing AGRANOMICS1 CEL files. The new CDFs can be accessed here.

First of all, the authors gave an overview of the AGRONOMICS1 tiling array. It contains all the probes from the traditionally used Affymetrix ATH1 array, but has additional probes which mean the AGRONOMICS1 array yields expression data for over 7000 more genes, around a third of the genome. 90% of annotated genes on the TAIR9 database are on the array. Mitochondrial and chloroplast genomes are completely represented, and sRNA, tRNA and miRNA can also be detected. The AGRONOMICS1 array has probes that represent both strands of the entire Arabidopsis genome, allowing epigenetic profiling. The quality is comparable to that of the ATH1 array.

This bumper GARNet Research Roundup begins with two sets of papers in related areas. First are three papers that investigate the biology of plasmodesmatata. These include work from the Faulkner lab at the JIC, the Band lab at Nottingham and a broad European collaboration that includes co-authors from Durham, Cambridge and St Andrews. The second[…]

Naresh Loudya from Royal Holloway University of London discusses a recent paper published in the Philosophical Transactions of the Royal Society B entitled ‘Retrograde signalling in a virescent mutant triggers an anterograde delay of chloroplast biogenesis that requires GUN1 and is essential for survival‘. Apologies for the high background noise early in the recording. http://blog.garnetcommunity.org.uk/wp-content/uploads/2020/05/Loudya_200520_edita-19052020-12.25.mp3Podcast:[…]

This edition of GARNet Research Roundup begins with two studies from the John Innes Centre. The first takes a detailed look at meiosis in Arabidopsis arenosa and the second introduces a novel mode of auxin perception. The third paper from the Grierson lab in Bristol uses innovative methods to assess root-soil cohesion through study of[…]

Andre Kuhn works with Lars Ostergaard at the John Innes Centre and discusses a recent Elife paper entitled ‘Direct ETTIN-auxin interaction controls chromatin states in gynoecium development‘. We discuss a new paradigm for auxin perception in the control gene expression. http://blog.garnetcommunity.org.uk/wp-content/uploads/2020/04/200420_Kuhn_edit-20042020-13.57.mp3Podcast: Play in new window | DownloadSubscribe: iTunes | Android | RSS

Bethany Eldridge and Tom Denbigh are co-first authors on a recent paper in Communications Biology entitled ‘Micro-scale interactions between Arabidopsis root hairs and soil particles influence soil erosion‘. We discuss the innovative experimental procedures that they designed to measure the interaction of root hairs with their environment! http://blog.garnetcommunity.org.uk/wp-content/uploads/2020/04/2004254_Eldridge_edit-24042020-11.16.mp3Podcast: Play in new window | DownloadSubscribe: iTunes[…]

GARNet conducted a short survey to assess community-interest in an UK-focused webinar series that will highlights the excellence in UK plant science. Approximately 100 respondants were supportive of the idea so we are kicking off the GARNet-Presents Webinar series on May 5th 2020. This series owes a huge debt of thanks to the widely popular[…]

Chris Morgan who works at the John Innes Centre discusses a recent PNAS paper entitled ‘Derived alleles of two axis proteins affect meiotic traits in autotetraploid Arabidopsis arenosa‘. We discuss the technical challenges of this research as well as the difficulties working from home (or not) with a super-resolution microscope! http://blog.garnetcommunity.org.uk/wp-content/uploads/2020/04/Morgan_200417_edit-17042020-14.59.mp3Podcast: Play in new window[…]

The use of automatic image analysis in the biological sciences has increased significantly in recent years, especially with automated image capture and the rise of phenotyping. This online course will help improve your understanding of image analysis methods, and improve your practical skills and ability to apply the techniques to your images. You will explore[…]

This Easter edition of the GARNet Research Roundup begins with research from Aberystwyth University that has developed a system for studying self-incompatability in self-compatible Arabidopsis. Next is an outstanding community-focussed study led from the John Innes Centre that outlines the development of new resources that better enable discovery-led science to be conducted within hexaploid wheat.[…]

Ludi Wang and Maurice Bosch work at Aberystwyth University and talk to the GARNet Community podcast about a recent paper in JXBot entitled ‘New opportunities and insights into Papaver self-incompatibility by imaging engineered Arabidopsis pollen‘. We discuss the challenges of live imaging Arabidopsis pollen tubes growing in liquid media!! http://blog.garnetcommunity.org.uk/wp-content/uploads/2020/04/Bosch_Wang_200406_edit-07042020-08.56.mp3Podcast: Play in new window |[…]